Aquisition of a confocal cryo-light microscope for correlative light and electron microscopy

项目总结： 原则调查员(PI)正在寻求行政补充，以获得共同焦点 低温相关光学/电子显微镜(CRYO-CLEM)的研究项目 细菌(R01GM141659)。本研究旨在了解这种相互作用的分子机制 在单链RNA噬菌体(单链RNA噬菌体)和它们的宿主细菌之间，重点是PP7 AP205噬菌体，分别侵染铜绿假单胞菌和不动杆菌。私人侦探希望 利用这些信息开发新的方法来对抗多重耐药细菌感染 减轻这些病原体的毒力。 该团队计划研究原位噬菌体包装过程，噬菌体吸附途径，以及 使用多尺度成像工具，包括荧光显微镜和 冷冻电子显微镜/断层扫描(Cryo-EM/ET)。他们已经获得了高分辨率的低温电磁 噬菌体PP7和AP205的单独或与宿主受体IV型菌毛的复合体结构，并具有 描述了与噬菌体感染时菌毛脱落相关的分子事件。 PI需要共焦冷冻-Clem，这将增强他们的成像能力和好处 他们的项目主要有两种方式。首先，低温Clem既能提供高分辨率成像，又能提供实时 该仪器具有良好的观察能力，弥补了传统光学显微镜和低温电子显微镜/电子显微镜之间的差距。其次， 该团队的目标是在假单胞菌生物膜和共聚焦荧光显微镜的背景下测试他们的发现 对于了解细菌细胞和噬菌体在感染过程中的空间组织是必不可少的。 张博士和曾博士，这对PI，已经与尼康和Linkam合作创建了一个冷冻-克莱姆装置 为他们的项目量身定做。仪器将放置在张博士新装修的实验室里，就在隔壁 曾博士的实验室在生物化学和生物物理系大楼。曾博士，他有超过15年的 有使用荧光显微镜的经验，将监督共焦的操作和维护 冷冻-克莱姆，与张博士合作。这一工具不仅将支持PIS目前的研究 该项目不仅对德克萨斯州农工行业的未来研究具有长期价值。

Project Summary: The Principle Investigators (PIs) are seeking an administrative supplement to obtain a confocal cryo-correlative light/electron microscope (cryo-CLEM) for their research project, "RNA Penetrations into Bacteria" (R01GM141659). The study aims to understand the molecular mechanisms underlying the interaction between single-stranded RNA bacteriophages (ssRNA phages) and their host bacteria, with a focus on PP7 and AP205 phages, which infect Pseudomonas aeruginosa and Acinetobacter spp., respectively. The PIs hope to use this information to develop new approaches to combat multi-drug-resistant bacterial infections by mitigating the virulence of these pathogens. The team plans to investigate the in situ phage packaging process, the phage adsorption pathway, and the RNA entry/pili detachment events using multi-scale imaging tools, including fluorescence microscopy and cryo-electron microscopy/tomography (cryo-EM/ET). They have already obtained high-resolution cryo-EM structures of both phage PP7 and AP205 alone or in complex with the host receptors, the type IV pili, and have characterized molecular events associated with pili detachment upon phage infection. The PIs request a confocal cryo-CLEM, which would enhance their imaging capabilities and benefit their project in two main ways. Firstly, a cryo-CLEM can provide both high-resolution imaging and real-time observation capabilities, bridging the gap between traditional light microscopy and cryo-EM/ET. Secondly, the team aims to test their findings in the context of Pseudomonas biofilms, and confocal fluorescence microscopy is essential for understanding the spatial organization of bacterial cells and phages during infection. Drs. Zhang and Zeng, the PIs, have collaborated with Nikon and Linkam to create a cryo-CLEM setup tailored to their project. The instrument will be located in Dr. Zhang's newly renovated lab, which is right next to Dr. Zeng's lab in the Department of Biochemistry and Biophysics building. Dr. Zeng, who has over 15 years of experience with fluorescence light microscopy, will supervise the operation and maintenance of the confocal cryo-CLEM in coordination with Dr. Zhang. This instrument will not only support the PIs’ current research project but also have long-term value for future research in the Texas A&M community.